The obligate intracellular bacterial pathogen Chlamydia trachomatis infects the ocular and genital epithelium to cause diseases of significant clinical importance. C. trachomatis modulates host cellular functions by translocating effector proteins across host and inclusion membranes. The lack of tools to perform classical molecular genetic analysis in Chlamydia, has hampered progress in identifying and characterizing effector proteins. However, given the small size of the its genome, expression-based functional approaches can be brought to genomic scale and significantly accelerate research in Chlamydia pathogenesis. Here, we propose to develop functional genomic tools to identify and characterize effector proteins and the dynamics of effector protein export during chlamydial infection. In our first aim, we propose to generate comprehensive yeast and mammalian expression libraries to systematically survey chlamydial proteins for functions related to their pathogenicity and apply immunological-based screens to identify novel secreted chlamydial proteins. These findings will be merged with proteomic data of Chlamydia elementary bodies to identify and characterize bacterial factors mediating entry and biogenesis of the nascent pathogenic vacuole. In our second aim, we extend the application of functional tools to characterize the chlamydial Type III secretion (TTS) system, the main portal for the delivery of effector proteins. Through two-hybrid technologies and biochemical approaches we propose to construct a protein-protein interaction map of this ancestral TTS apparatus to identify non-conserved components of the core translocon, needle complex and putative regulatory subunits. In particular, we will focus our studies on Chlamydia-specific proteins that interact with the basal structure of the TSS system, including a novel TTS chaperone we have identified and its cognate cargo.